Method of treating cancer associated with a RAS mutation

ABSTRACT

Disclosed herein are methods of treating a cancer characterized by expressing a mutant form of a RAS protein. Some embodiments relate to treatment of cancer by administering Plinabulin to a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/555,963, filed on Sep. 5, 2017, which was the National Phase ofInternational Application No. PCT/US2016/020390, filed on Mar. 2, 2016and published on Sep. 15, 2016 as WO 2016/144635, which claims thebenefit of U.S. Provisional Application No. 62/129,654, filed on Mar. 6,2015, and U.S. Provisional Application No. 62/249,788, filed on Nov. 2,2015, the disclosures of which are incorporated herein by reference intheir entireties.

BACKGROUND Field

The present invention relates to the field of chemistry and medicine.More particularly, the present invention relates to method of treatingcancer associated with a RAS mutation using Plinabulin.

Description of the Related Art

RAS proteins function as binary molecular switches that controlintracellular signaling networks. RAS-regulated signal pathways controlprocesses such as actin cytoskeletal integrity, proliferation,differentiation, cell adhesion, apoptosis, and cell migration. The RASsubfamily includes at least 21 members such as KRAS, NRAS, and HRAS. RASor RAS-related protein relay signals from outside the cell to the cell'snucleus. These signals instruct the cell to grow and divide or to matureand take on specialized functions (differentiate). The RAS proteinsbelong to a class of protein called GTPase, which means it converts amolecule called GTP into another molecule called GDP. A RAS protein actslike a switch, and it is turned on and off by the GTP and GDP molecules.To transmit signals, the RAS protein must be turned on by attaching(binding) to a molecule of GTP. A RAS protein is turned off(inactivated) when it converts the GTP to GDP. When the protein is boundto GDP, it does not relay signals to the cell's nucleus. When a RASprotein is mutated at certain codons, the RAS protein is stuck in itsGTP bound state, so it keeps sending signal to nucleus and the cellcould not stop dividing, which is un-controlled cell growth or cancer.

The RAS genes belong to a class of genes known as oncogenes. A mutantform of RAS gene has the potential to cause normal cells to becomecancerous. The proteins produced from these RAS genes are GTPases. Theseproteins play important roles in cell division, cell differentiation,and the cell apoptosis. However, the mechanisms by which oncogenic RAScoordinates the shift in metabolism to sustain tumor growth,particularly in the tumor microenvironment, and whether specificmetabolic pathways are essential for RAS-mediated tumor maintenanceremain areas of active investigation. Cancers characterized by a RASmutation are difficult to treat using known therapies, and there are noapproved drugs for treating these cancers. RAS mutation is a negativepredictor of cancer patient survival; cancer patients have much poorerprognostic outcome (or shorter overall survival) when their cancer hasRAS mutation. Thus, there is a dire unmet need for a more effectivetreatment for cancer associated with oncogenic RAS gene mutation.

SUMMARY

Some embodiments relate to a method of treating a cancer characterizedby expressing a mutant form of RAS protein, comprising administeringPlinabulin to a subject in need thereof.

Some embodiments relate to a method of treating a cancer characterizedby expressing a mutant form of KRAS protein, comprising administeringPlinabulin to a subject in need thereof.

Some embodiments relate to a method of treating a cancer characterizedby expressing a mutant form of NRAS protein, comprising administeringPlinabulin to a subject in need thereof.

Some embodiments relate to a method of inhibiting proliferation of acell having a RAS mutation, comprising contacting the cell withPlinabulin.

Some embodiments relate to a method of inducing apoptosis in a cellhaving a RAS mutation, comprising contacting the cell with Plinabulin.

Some embodiments relate to a method of inhibiting progression of acancer characterized by expressing a mutant form of RAS protein in asubject, comprising administering Plinabulin to a subject in needthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1d show the proneural genetically engineered murine model(GEMM) of glioblastoma (GBM) that mimics human pathology. FIG. 1a showsT2 MRI images of a human GBM that display peritumoral edema; and FIG. 1bshows T2 MRI images of mouse GBM that display peritumoral edema; FIG. 1cshows human micrograph images of H&E stains of a GBM showing hallmarkpseudopalisading necrosis and microvascular proliferation; FIG. 1d showsmouse micrograph images of H&E stains of a GBM showing hallmarkpseudopalisading necrosis and microvascular proliferation.

FIG. 2 shows tumor size of mice with PDGF-induced gliomas treated withvehicle, temozolomide or fractionated radiation.

FIG. 3 shows the tumor weight autopsy in mice treated with vehicle only,Docetaxel only, and a combination of Plinabulin and Docetaxel.

FIG. 4 shows the survival rate of mice with PDGF-induced gliomascharacterized by expression of KRAS mutation that were treated withcontrol and Plinabulin.

FIG. 5 shows the survival rate of mice with PDGF-induced gliomascharacterized by expression of KRAS mutation that were treated with thecombination of plinabulin, temozolomide, and radiation and thecombination of temozolomide and radiation.

FIG. 6 shows the change in tumor volume in mice with the HCT-15 (Krasmutation G13D) human colon tumor xenograft throughout the course of thestudy in mice treated with vehicle control, plinabulin, irinotecan, orthe combination of plinabulin and irinotecan.

FIG. 7 shows the change in tumor volume in mice with the Lovo (Krasmutation, p.G13D) human colon tumor xenograft throughout the course ofthe study in mice treated with vehicle control, plinabulin, irinotecan,or the combination of plinabulin and irinotecan.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Plinabulin,(3Z,6Z)-3-Benzylidene-6-{[5-(2-methyl-2-propanyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione,is a synthetic analog of the natural compound phenylahistin. Plinabulincan be readily prepared according to methods and procedures detailed inU.S. Pat. Nos. 7,064,201 and 7,919,497, which are incorporated herein byreference in their entireties. Some embodiments relate to the use ofPlinabulin to treat cancer associated with an oncogenic RAS mutation.Some embodiments relate to the use of Plinabulin to treat a cancercharacterized by expressing a mutant form of RAS protein in a subject.Some embodiments relate to the use of Plinabulin to inhibitproliferation of a cell having a RAS mutation. Some embodiments relateto the use of Plinabulin to induce apoptosis in a cell having a RASmutation. Some embodiments relate to the use of Plinabulin to inhibitprogression of a cancer that is characterized by expressing a mutantform of RAS protein in a subject. In some embodiments, the RAS proteinis a KRAS protein. In some embodiments, the RAS protein is a NRASprotein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications, and other publications are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

The term “mammal” is used in its usual biological sense. Thus, itspecifically includes, but is not limited to, primates, includingsimians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep,goats, swine, rabbits, dogs, cats, rodents, rats, mice guinea pigs, orthe like.

An “effective amount” or a “therapeutically effective amount” as usedherein refers to an amount of a therapeutic agent that is effective torelieve, to some extent, or to reduce the likelihood of onset of, one ormore of the symptoms of a disease or condition, and includes curing adisease or condition.

“Treat,” “treatment,” or “treating,” as used herein refers toadministering a compound or pharmaceutical composition to a subject forprophylactic and/or therapeutic purposes. The term “prophylactictreatment” refers to treating a subject who does not yet exhibitsymptoms of a disease or condition, but who is susceptible to, orotherwise at risk of, a particular disease or condition, whereby thetreatment reduces the likelihood that the patient will develop thedisease or condition. The term “therapeutic treatment” refers toadministering treatment to a subject already suffering from a disease orcondition.

The term “pharmaceutically acceptable salt” refers to salts that retainthe biological effectiveness and properties of a compound and, which arenot biologically or otherwise undesirable for use in a pharmaceutical.In many cases, the compounds disclosed herein are capable of formingacid and/or base salts by virtue of the presence of amino and/orcarboxyl groups or groups similar thereto. Pharmaceutically acceptableacid addition salts can be formed with inorganic acids and organicacids. Inorganic acids from which salts can be derived include, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid, phosphoric acid, and the like. Organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceuticallyacceptable salts can also be formed using inorganic and organic bases.Inorganic bases from which salts can be derived include, for example,bases that contain sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum, and the like;particularly preferred are the ammonium, potassium, sodium, calcium andmagnesium salts. In some embodiments, treatment of the compoundsdisclosed herein with an inorganic base results in loss of a labilehydrogen from the compound to afford the salt form including aninorganic cation such as Li⁺, Na⁺, K⁺, Mg²⁺ and Ca²⁺ and the like.Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like, specifically such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. Many such salts are known in the art, as described in WO87/05297, Johnston et al., published Sep. 11, 1987 (incorporated byreference herein in its entirety).

In some embodiments, the composition can further include one or morepharmaceutically acceptable diluents. In some embodiments, thepharmaceutically acceptable diluent can include Kolliphor® HS15(Polyethylene glycol (15)-hydroxystearate). In some embodiments, thepharmaceutically acceptable diluent can include propylene glycol. Insome embodiments, the pharmaceutically acceptable diluents can includeKolliphor® HS15 and propylene glycol. In some embodiments, thepharmaceutically acceptable diluents can include Kolliphor® HS15 andpropylene glycol, wherein the Kolliphor® HS15 is about 40% by weight andpropylene glycol is about 60% by weight based on the total weight of thediluents. In some embodiments, the composition can further include oneor more other pharmaceutically acceptable excipients.

Standard pharmaceutical formulation techniques can be used to make thepharmaceutical compositions described herein, such as those disclosed inRemington's The Science and Practice of Pharmacy, 21st Ed., LippincottWilliams & Wilkins (2005), incorporated herein by reference in itsentirety. Accordingly, some embodiments include pharmaceuticalcompositions comprising: (a) a safe and therapeutically effective amountof Plinabulin or pharmaceutically acceptable salts thereof; and (b) apharmaceutically acceptable carrier, diluent, excipient or combinationthereof.

Other embodiments include co-administering Plinabulin and an additionaltherapeutic agent in separate compositions or the same composition.Thus, some embodiments include a first pharmaceutical compositioncomprising: (a) a safe and therapeutically effective amount ofPlinabulin or pharmaceutically acceptable salts thereof and (b) apharmaceutically acceptable carrier, diluent, excipient or combinationthereof; and a second pharmaceutical composition comprising: (a) a safeand therapeutically effective amount of an additional therapeutic agentand (b) a pharmaceutically acceptable carrier, diluent, excipient orcombination thereof. Some embodiments include a pharmaceuticalcomposition comprising: (a) a safe and therapeutically effective amountof Plinabulin or pharmaceutically acceptable salts thereof; (b) a safeand therapeutically effective amount of an additional therapeutic agent;and (c) a pharmaceutically acceptable carrier, diluent, excipient orcombination thereof.

Administration of the pharmaceutical compositions described herein canbe via any of the accepted modes of administration for agents that servesimilar utilities including, but not limited to, orally, sublingually,buccally, subcutaneously, intravenously, intranasally, topically,transdermally, intradermally, intraperitoneally, intramuscularly,intrapulmonarilly, vaginally, rectally, or intraocularly. Oral andparenteral administrations are customary in treating the indicationsthat are the subject of the preferred embodiments.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. In addition, various adjuvants such as are commonly usedin the art may be included. Considerations for the inclusion of variouscomponents in pharmaceutical compositions are described, e.g., in Gilmanet al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis ofTherapeutics, 8th Ed., Pergamon Press, which is incorporated herein byreference in its entirety.

Some examples of substances, which can serve aspharmaceutically-acceptable carriers or components thereof, are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerin, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe TWEENS; wetting agents, such sodium lauryl sulfate; coloring agents;flavoring agents; tableting agents, stabilizers; antioxidants;preservatives; pyrogen-free water; isotonic saline; and phosphate buffersolutions.

The compositions described herein are preferably provided in unit dosageform. As used herein, a “unit dosage form” is a composition containingan amount of a compound or composition that is suitable foradministration to an animal, preferably a mammalian subject, in a singledose, according to good medical practice. The preparation of a single orunit dosage form however, does not imply that the dosage form isadministered once per day or once per course of therapy. Such dosageforms are contemplated to be administered once, twice, thrice or moreper day and may be administered as infusion over a period of time (e.g.,from about 30 minutes to about 2-6 hours), or administered as acontinuous infusion, and may be given more than once during a course oftherapy, although a single administration is not specifically excluded.The skilled artisan will recognize that the formulation does notspecifically contemplate the entire course of therapy and such decisionsare left for those skilled in the art of treatment rather thanformulation.

The compositions useful as described above may be in any of a variety ofsuitable forms for a variety of routes for administration, for example,for oral, sublingual, buccal, nasal, rectal, topical (includingtransdermal and intradermal), ocular, intracerebral, intracranial,intrathecal, intra-arterial, intravenous, intramuscular, or otherparental routes of administration. The skilled artisan will appreciatethat oral and nasal compositions include compositions that areadministered by inhalation, and made using available methodologies.Depending upon the particular route of administration desired, a varietyof pharmaceutically-acceptable carriers well-known in the art may beused. Pharmaceutically-acceptable carriers include, for example, solidor liquid fillers, diluents, hydrotropies, surface-active agents, andencapsulating substances. Optional pharmaceutically-active materials maybe included, which do not substantially interfere with the activity ofthe compound or composition. The amount of carrier employed inconjunction with the compound or composition is sufficient to provide apractical quantity of material for administration per unit dose of thecompound. Techniques and compositions for making dosage forms useful inthe methods described herein are described in the following references,all incorporated by reference herein: Modern Pharmaceutics, 4th Ed.,Chapters 9 and 10 (Banker & Rhodes, editors, 2002); Lieberman et al.,Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction toPharmaceutical Dosage Forms 8th Edition (2004).

Various oral dosage forms can be used, including such solid forms astablets, capsules (e.g., liquid gel capsule and solid gel capsule),granules and bulk powders. Tablets can be compressed, tablet triturates,enteric-coated, sugar-coated, film-coated, or multiple-compressed,containing suitable binders, lubricants, diluents, disintegratingagents, coloring agents, flavoring agents, flow-inducing agents, andmelting agents. Liquid oral dosage forms include aqueous solutions,emulsions, suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules, and effervescent preparations reconstitutedfrom effervescent granules, containing suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, meltingagents, coloring agents and flavoring agents.

The pharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration is well-known in the art.Tablets typically comprise conventional pharmaceutically-compatibleadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. The selection of carriercomponents depends on secondary considerations like taste, cost, andshelf stability, which are not critical, and can be readily made by aperson skilled in the art.

Peroral compositions also include liquid solutions, emulsions,suspensions, and the like. The pharmaceutically-acceptable carrierssuitable for preparation of such compositions are well known in the art.Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, AVICEL RC-591, tragacanth and sodium alginate; typicalwetting agents include lecithin and polysorbate 80; and typicalpreservatives include methyl paraben and sodium benzoate. Peroral liquidcompositions may also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

Such compositions may also be coated by conventional methods, typicallywith pH or time-dependent coatings, such that the subject composition isreleased in the gastrointestinal tract in the vicinity of the desiredtopical application, or at various times to extend the desired action.Such dosage forms typically include, but are not limited to, one or moreof cellulose acetate phthalate, polyvinylacetate phthalate,hydroxypropyl methyl cellulose phthalate, ethyl cellulose, Eudragitcoatings, waxes and shellac.

Compositions described herein may optionally include other drug actives.

Other compositions useful for attaining systemic delivery of the subjectcompounds include sublingual, buccal and nasal dosage forms. Suchcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol; and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose and hydroxypropylmethyl cellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

A liquid composition, which is formulated for topical ophthalmic use, isformulated such that it can be administered topically to the eye. Thecomfort may be maximized as much as possible, although sometimesformulation considerations (e.g. drug stability) may necessitate lessthan optimal comfort. In the case that comfort cannot be maximized, theliquid may be formulated such that the liquid is tolerable to thepatient for topical ophthalmic use. Additionally, an ophthalmicallyacceptable liquid may either be packaged for single use, or contain apreservative to prevent contamination over multiple uses.

For ophthalmic application, solutions or medicaments are often preparedusing a physiological saline solution as a major vehicle. Ophthalmicsolutions may preferably be maintained at a comfortable pH with anappropriate buffer system. The formulations may also containconventional, pharmaceutically acceptable preservatives, stabilizers andsurfactants.

Preservatives that may be used in the pharmaceutical compositionsdisclosed herein include, but are not limited to, benzalkonium chloride,PHMB, chlorobutanol, thimerosal, phenylmercuric, acetate andphenylmercuric nitrate. A useful surfactant is, for example, Tween 80.Likewise, various useful vehicles may be used in the ophthalmicpreparations disclosed herein. These vehicles include, but are notlimited to, polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose and purifiedwater.

Tonicity adjustors may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjustor.

Various buffers and means for adjusting pH may be used so long as theresulting preparation is ophthalmically acceptable. For manycompositions, the pH will be between 4 and 9. Accordingly, buffersinclude acetate buffers, citrate buffers, phosphate buffers and boratebuffers. Acids or bases may be used to adjust the pH of theseformulations as needed.

Ophthalmically acceptable antioxidants include, but are not limited to,sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole and butylated hydroxytoluene.

Other excipient components, which may be included in the ophthalmicpreparations, are chelating agents. A useful chelating agent is edetatedisodium (EDTA), although other chelating agents may also be used inplace or in conjunction with it.

For topical use, creams, ointments, gels, solutions or suspensions,etc., containing the composition disclosed herein are employed. Topicalformulations may generally be comprised of a pharmaceutical carrier,co-solvent, emulsifier, penetration enhancer, preservative system, andemollient.

For intravenous administration, the compositions described herein may bedissolved or dispersed in a pharmaceutically acceptable diluent, such asa saline or dextrose solution. Suitable excipients may be included toachieve the desired pH, including but not limited to NaOH, sodiumcarbonate, sodium acetate, HCl, and citric acid. In various embodiments,the pH of the final composition ranges from 2 to 8, or preferably from 4to 7. Antioxidant excipients may include sodium bisulfite, acetonesodium bisulfite, sodium formaldehyde, sulfoxylate, thiourea, and EDTA.Other non-limiting examples of suitable excipients found in the finalintravenous composition may include sodium or potassium phosphates,citric acid, tartaric acid, gelatin, and carbohydrates such as dextrose,mannitol, and dextran. Further acceptable excipients are described inPowell, et al., Compendium of Excipients for Parenteral Formulations,PDA J Pharm Sci and Tech 1998, 52 238-311 and Nema et al., Excipientsand Their Role in Approved Injectable Products: Current Usage and FutureDirections, PDA J Pharm Sci and Tech 2011, 65 287-332, both of which areincorporated herein by reference in their entirety. Antimicrobial agentsmay also be included to achieve a bacteriostatic or fungistaticsolution, including but not limited to phenylmercuric nitrate,thimerosal, benzethonium chloride, benzalkonium chloride, phenol,cresol, and chlorobutanol.

The compositions for intravenous administration may be provided tocaregivers in the form of one or more solids that are reconstituted witha suitable diluent such as sterile water, saline or dextrose in watershortly prior to administration. In other embodiments, the compositionsare provided in solution ready to administer parenterally. In stillother embodiments, the compositions are provided in a solution that isfurther diluted prior to administration. In embodiments that includeadministering a combination of a compound described herein and anotheragent, the combination may be provided to caregivers as a mixture, orthe caregivers may mix the two agents prior to administration, or thetwo agents may be administered separately.

The actual dose of the active compounds described herein depends on thespecific compound, and on the condition to be treated; the selection ofthe appropriate dose is well within the knowledge of the skilledartisan. In some embodiments, a single dose of Plinabulin or othertherapeutic agent may be from about 5 mg/m² to about 150 mg/m² of bodysurface area, from about 5 mg/m² to about 100 mg/m² of body surfacearea, from about 10 mg/m² to about 100 mg/m² of body surface area, fromabout 10 mg/m² to about 80 mg/m² of body surface area, from about 10mg/m² to about 50 mg/m² of body surface area, from about 10 mg/m² toabout 40 mg/m² of body surface area, from about 10 mg/m² to about 30mg/m² of body surface area, from about 13.5 mg/m² to about 100 mg/m² ofbody surface area, from about 13.5 mg/m² to about 80 mg/m² of bodysurface area, from about 13.5 mg/m² to about 50 mg/m² of body surfacearea, from about 13.5 mg/m² to about 40 mg/m² of body surface area, fromabout 13.5 mg/m² to about 30 mg/m² of body surface area, from about 15mg/m² to about 80 mg/m² of body surface area, from about 15 mg/m² toabout 50 mg/m² of body surface area, or from about 15 mg/m² to about 30mg/m² of body surface area. In some embodiments, a single dose ofPlinabulin or other therapeutic agent may be from about 13.5 mg/m² toabout 30 mg/m² of body surface area. In some embodiments, a single doseof Plinabulin or other therapeutic agent may be about 5 mg/m², about 10mg/m², about 12.5 mg/m², about 13.5 mg/m², about 15 mg/m², about 17.5mg/m², about 20 mg/m², about 22.5 mg/m², about 25 mg/m², about 27.5mg/m², about 30 mg/m², about 40 mg/m², about 50 mg/m², about 60 mg/m²,about 70 mg/m², about 80 mg/m², about 90 mg/m², or about 100 mg/m², ofbody surface area.

In some embodiments, a single dose of Plinabulin or other therapeuticagent may be from about 5 mg to about 300 mg, from about 5 mg to about200 mg, from about 7.5 mg to about 200 mg, from about 10 mg to about 100mg, from about 15 mg to about 100 mg, from about 20 mg to about 100 mg,from about 30 mg to about 100 mg, from about 40 mg to about 100 mg, fromabout 10 mg to about 80 mg, from about 15 mg to about 80 mg, from about20 mg to about 80 mg, from about 30 mg to about 80 mg, from about 40 mgto about 80 mg, from about 10 mg to about 60 mg, from about 15 mg toabout 60 mg, from about 20 mg to about 60 mg, from about 30 mg to about60 mg, or from about 40 mg to about 60 mg, In some embodiments, a singledose of Plinabulin or other therapeutic agent may be from about 20 mg toabout 60 mg, from about 27 mg to about 60 mg, from about 20 mg to about45 mg, or from about 27 mg to about 45 mg. In some embodiments, a singledose of Plinabulin or other therapeutic agent may be about 5 mg, about10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg,about 100 mg, about 125 mg, about 150 mg, or about 200 mg.

The administration period can be a multi-week treatment cycle as long asthe tumor remains under control and the regimen is clinically tolerated.In some embodiments, a single dosage of Plinabulin or other therapeuticagent can be administered once a week, and preferably once on each ofday 1 and day 8 of a three-week (21 day) treatment cycle. In someembodiments, a single dosage of Plinabulin or other therapeutic agentcan be administered once a week, twice a week, three times per week,four times per week, five times per week, six times per week, or dailyduring a one-week, two-week, three-week, four-week, or five-weektreatment cycle. The administration can be on the same or different dayof each week in the treatment cycle.

The treatment cycle can be repeated as long as the regimen is clinicallytolerated. In some embodiments, the treatment cycle is repeated for ntimes, wherein n is an integer in the range of 2 to 30. In someembodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, anew treatment cycle can occur immediately after the completion of theprevious treatment cycle. In some embodiments, a new treatment cycle canoccur a period of time after the completion of the previous treatmentcycle.

In some embodiments, the compositions described herein can be used incombination with other therapeutic agents. In some embodiments, thecompositions described herein can be administered or used in combinationwith treatments such as chemotherapy, radiation, and biologic therapies.

Methods of Treatment

Some embodiments relate to a method of treating a cancer characterizedby expressing a mutant form of a RAS protein, comprising administeringPlinabulin to a subject in need thereof. Some embodiments relate to amethod of treating a cancer associated with an oncogenic RAS mutation,comprising administering Plinabulin to a subject in need thereof.

In some embodiments, the mutant form of the RAS protein is a mutant formof a KRAS, NRAS, or HRAS protein. In some embodiments, the RAS proteinis a KRAS, NRAS, or HRAS protein. In some embodiments, the mutant formof the RAS is a mutant form of the KRAS protein. In some embodiments,the mutant form of the RAS is a mutant form of the NRAS protein. In someembodiments, the RAS gene mutation is KRAS gene mutation. In someembodiments, the RAS gene mutation is NRAS gene mutation.

Some embodiments relate to a method of treating a cancer characterizedby expressing a mutant form of a KRAS protein, comprising administeringPlinabulin to a subject in need thereof. Some embodiments relate to amethod of treating a cancer associated with an oncogenic KRAS mutation,comprising administering Plinabulin to a subject in need thereof.

Some embodiments relate to a method of treating a cancer characterizedby expressing a mutant form of a NRAS protein, comprising administeringPlinabulin to a subject in need thereof. Some embodiments relate to amethod of treating a cancer associated with an oncogenic NRAS mutation,comprising administering Plinabulin to a subject in need thereof.

Some embodiments of the present invention include methods of treatingcancer with Plinabulin and compositions comprising Plinabulin describedherein. Some methods include administering a compound, composition,pharmaceutical composition described herein to a subject in needthereof. In some embodiments, a subject can be an animal, e.g., amammal, a human.

In some embodiments, cancer is selected from colorectal cancer,pancreatic cancer, renal cancer, lung cancer, liver cancer, breastcancer, prostate cancer, gastrointestinal cancer, peritoneal cancer,melanoma, endometrial cancer, ovarian cancer, cervical cancer, uterinecarcinoma, bladder cancer, glioblastoma, brain metastases, salivarygland carcinoma, thyroid cancer, brain cancer, lymphoma, myeloma, andhead and neck cancer. In some embodiments, the cancer is selected fromsquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,hepatocellular carcinoma, colon cancer, endometrial carcinoma, andhepatocellular carcinoma. In some embodiments, the cancer is selectedfrom colorectal cancer, prostate cancer, breast cancer, lung cancer,endometrial cancer, multiple myeloma, pancreatic, renal cancer, andglioblastoma. In some embodiments, the cancer is selected from non-smallcell lung cancer, pancreatic cancer, and glioblastoma. In someembodiments, the cancer is non-small cell lung cancer.

In some embodiments, the KRAS comprises mutation at one or morepositions selected from codons 12, 13, 59, and 61. In some embodiments,the mutant form of the KRAS protein has mutation at one or more aminoacid positions selected from G12, G13, S17, P34, A59, and Q61. In someembodiments, the mutant form of the KRAS protein has one or more aminoacid substitutions selected from the group consisting of G12C, G12S,G12R, G12F, G12L, G12N, G12A, G12D, G12V, G13C, G13S, G13D, G13V, G13P,S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, and Q61H. In someembodiments, the mutant form of the KRAS protein has mutation at one ormore amino acid positions selected from G12, G13, A59, Q61, K117 andA146. In some embodiments, the mutant form of the KRAS protein has oneor more amino acid substitutions selected from the group consisting ofG12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V,A59E, A59G, A59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, A146P,A146T and A146V. In some embodiments, the mutant form of the KRASprotein has mutation at one or more amino acid positions selected fromof G12, G13, A59 and Q61. In some embodiments, the mutant form of theKRAS protein has one or more amino acid substitutions selected from thegroup consisting of G12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R,G13S, G13A, G13D, A59E, A59G, A59T, Q61K, Q61L, Q61R and Q61H. In someembodiments, the mutant form of the KRAS protein has mutation at one ormore amino acid positions selected from G12, G13, and D153. In someembodiments, the mutant form of the KRAS protein has one or more aminoacid substitutions selected from the group consisting of G12A, G12C,G12D, G12V, G12S, G13D, and D153V. In some embodiments, the mutant formof the KRAS protein has one or more amino acid substitutions selectedfrom G12C, G12S, and D153V.

In some embodiments, the NRAS comprises mutation at one or morepositions selected from codons 12, 13, 59, 61, and 146. In someembodiments, the mutant form of the KRAS protein has mutation at one ormore amino acid positions selected from G12, G13, A59, Q61, K117 andA146. In some embodiments, the mutant form of the KRAS protein has oneor more amino acid substitutions selected from the group consisting ofG12C, G12R, G12S, G12A, G12D, G12V, G13C, G13R, G13S, G13A, G13D, G13V,A59D, A59T, Q61K, Q61L, Q61R, Q61H, K117N, K117R, K117E, A146P, A146Tand A146V. In some embodiments, the mutant form of the NRAS protein hasmutation at one or more amino acid positions at Q61 or A146. In someembodiments, the mutant form of the NRAS protein has one or more aminoacid substitutions selected from Q61K, Q61H, Q61R, Q61L, Q61N, Q61E,Q61P, A146T, A146P, A146V, and any combinations thereof. In someembodiments, the mutant form of the NRAS protein has one or more aminoacid substitutions selected from Q61H, Q61R, Q61L, and any combinationsthereof.

Some embodiments relate to a method of inhibiting proliferation of acell having a RAS mutation, comprising administering Plinabulin to asubject in need thereof. Some embodiments relate to a method of inducingapoptosis in a cell having a RAS mutation, comprising contacting thecell with Plinabulin. In some embodiments, the contacting includesadministering Plinabulin to a subject in need thereof. Some embodimentsrelate to a method of inhibiting progression of a cancer characterizedby expressing a mutant form of RAS in a subject, comprising contactingthe cell with Plinabulin. In some embodiments, the contacting includesadministering Plinabulin to a subject in need thereof.

Some embodiments relate to a method of inhibiting proliferation of acell having a KRAS mutation, comprising administering Plinabulin to asubject in need thereof. Some embodiments relate to a method of inducingapoptosis in a cell having a KRAS mutation, comprising contacting thecell with Plinabulin. Some embodiments relate to a method of inhibitingprogression of a cancer characterized by expressing a mutant form ofKRAS in a subject, comprising contacting the cell with Plinabulin.

Some embodiments relate to a method of inhibiting proliferation of acell having a NRAS mutation, comprising administering Plinabulin to asubject in need thereof. Some embodiments relate to a method of inducingapoptosis in a cell having a NRAS mutation, comprising contacting thecell with Plinabulin. Some embodiments relate to a method of inhibitingprogression of a cancer characterized by expressing a mutant form ofNRAS in a subject, comprising contacting the cell with Plinabulin.

In some embodiments, the subject is a human.

Further embodiments include administering a combination of compounds toa subject in need thereof. A combination can include a compound,composition, pharmaceutical composition described herein with anadditional medicament.

Some embodiments include co-administering a compound, composition,and/or pharmaceutical composition described herein, with an additionalmedicament. By “co-administration,” it is meant that the two or moreagents may be found in the patient's bloodstream at the same time,regardless of when or how they are actually administered. In oneembodiment, the agents are administered simultaneously. In one suchembodiment, administration in combination is accomplished by combiningthe agents in a single dosage form. In another embodiment, the agentsare administered sequentially. In one embodiment the agents areadministered through the same route, such as orally. In anotherembodiment, the agents are administered through different routes, suchas one being administered orally and another being administered i.v.

The treatment method described herein can include co-administeringPlinabulin with an additional active agent. In some embodiments, themethod further includes administering an additional therapeutic agent.In some embodiments, the additional therapeutic agent is docetaxel andthe cancer is human non-small cell lung cancer. In some embodiments, theadditional therapeutic agent is Bevacizumab and the cancer is humannon-small cell lung cancer. In some embodiments, the additionaltherapeutic agent is Irinotecan and the cancer is colon cancer. In someembodiments, the additional therapeutic agent is Temolozomide and thecancer is glioblastoma. In some embodiments, the additional therapeuticagent is irinotecan and the cancer is colon cancer.

In some embodiments, the additional therapeutic agent can betemozolomide, bevicizumab, everolimus, carmustine, lomustine,procarbazine, vincristine, irinotecan, cisplatin, carboplatin,methatrexate, etoposide, vinblasatine, bleomycin, actinomycin,cyclophosphamide, or ifosfamide. In some embodiments, the additionaltherapeutic agent is selected from bevacizumab, docetaxel, Irinotecan,and temozolomide. In some embodiments, the additional therapeutic agentis docetaxel. In some embodiments, the additional therapeutic agent isirinotecan. In some embodiments, the additional therapeutic agent isbevacizumab. In some embodiments, the additional therapeutic agent istemozolomide. In some embodiments, the additional therapeutic agent isgemcitabine. In some embodiments, the additional therapeutic agent iscarmustine. In some embodiments, the additional therapeutic agent islomustine.

The treatment method described herein can also be used in combinationwith a radiation therapy.

The method described herein can further include identifying a patienthaving a cancer characterized by expressing a mutant type of RASprotein. In some embodiments, identifying a patient can includedetermining whether the patient has a RAS mutation. Some embodimentsrelate to a method for treating cancer in a patient identified as havinga RAS mutation, the method comprising administering to the patient apharmaceutically effective amount of Plinabulin, wherein the patient hasbeen identified by (i) collecting a sample from the patient; (ii)isolating DNA from the sample; (iii) amplifying a RAS gene or fragmentthereof in the isolated DNA; and (iv) detecting whether there is amutation in the amplified RAS gene, thereby determining whether thepatient has a cancer characterized by a RAS mutation. Examples ofmethods for detecting RAS mutation include but are not limited toAmplified Refractory Mutation System (ARMS) PCR, BEAMing assays, digitalPCR, and other suitable primers, and probes for sequencing or PCR.

The method described herein can further include identifying a patienthaving a cancer characterized by expressing a mutant type of KRAS. Insome embodiments, identifying a patient can include determining whetherthe patient has a KRAS mutation. Some embodiments relate to a method fortreating cancer in a patient identified as having a KRAS mutation, themethod comprising administering to the patient a pharmaceuticallyeffective amount of Plinabulin, wherein the patient has been identifiedby (i) collecting a sample from the patient; (ii) isolating DNA from thesample; (iii) amplifying a KRAS gene or fragment thereof in the isolatedDNA; and (iv) detecting whether there is a mutation in the amplifiedKRAS gene, thereby determining whether the patient has a cancercharacterized by a KRAS mutation.

The method described herein can further include testing for a KRASmutation. In some embodiments, KRAS mutations, e.g., at codons 12, 13,59, 61 and/or 146 may be detected from pathology samples taken from thepatient. In some embodiments of such testing, DNA is removed from thesample and tested against labeled oligonucleotide probes using PCR toamplify the targeted mutated DNA to permit detection. In someembodiments, commercial testing centers can carry out such tests. Insome embodiments, a test kit such as the TheraScreen: K-RAS Mutation kit(DxS Ltd, 48 Grafton Street, Manchester M1 3 9XX, UK) may be used. Forexample, the TheraScreen kit can detect mutations in codons 12 and 13 ofthe K-RAS oncogene:

GIy 12 Asp (GGT>GAT) GIy 12 Arg (GG1>CGT)

GIy 12 Ala (GGT>GCT) GIy 12Cy s (GGT>TGT)

GIy 12VaI (GGT>GTT) GIy 13 Asp (GGOGAC) Gly12Ser (GGT>AGT)

The method described herein can further include identifying a patienthaving a cancer characterized by expressing a mutant type of NRASprotein. In some embodiments, identifying a patient can includedetermining whether the patient has a NRAS mutation. Some embodimentsrelate to a method for treating cancer in a patient identified as havinga NRAS mutation, the method comprising administering to the patient apharmaceutically effective amount of Plinabulin, wherein the patient hasbeen identified by (i) collecting a sample from the patient; (ii)isolating DNA from the sample; (iii) amplifying a NRAS gene or fragmentthereof in the isolated DNA; and (iv) detecting whether there is amutation in the amplified NRAS gene, thereby determining whether thepatient has a cancer characterized by a NRAS mutation.

The method described herein can further include testing for a NRASmutation. In some embodiments, NRAS mutations, e.g., at codons 12, 13,59, 61 and/or 146, may be detected from pathology samples taken from thepatient. In some embodiments of such testing, DNA is removed from thesample and tested against labeled oligonucliotide probes using PCR toamplify the targeted mutated DNA to permit detection. Severalcommercially available kits (see Dxs Diagnostic Innovations, AppliedBiosystems, and Quest diagnostics), primers and probes for sequencing orPCR can be designed based on the codon mutations of NRAS.

The method described herein can further include identifying a patienthaving a cancer characterized by expressing a mutant type of HRASprotein. In some embodiments, identifying a patient can includedetermining whether the patient has a HRAS mutation. Some embodimentsrelate to a method for treating cancer in a patient identified as havinga HRAS mutation, the method comprising administering to the patient apharmaceutically effective amount of Plinabulin, wherein the patient hasbeen identified by (i) collecting a sample from the patient; (ii)isolating DNA from the sample; (iii) amplifying a HRAS gene or fragmentthereof in the isolated DNA; and (iv) detecting whether there is amutation in the amplified HRAS gene, thereby determining whether thepatient has a cancer characterized by a HRAS mutation.

To further illustrate this invention, the following examples areincluded. The examples should not, of course, be construed asspecifically limiting the invention. Variations of these examples withinthe scope of the claims are within the purview of one skilled in the artand are considered to fall within the scope of the invention asdescribed, and claimed herein. The reader will recognize that theskilled artisan, armed with the present disclosure, and skill in the artis able to prepare and use the invention without exhaustive examples.

EXAMPLES Example 1

All cell lines were grown in their respective proper growth mediasupplemented with 2-10% FBS and were housed in an atmosphere of 5% CO₂at 37° C.

Cells were plated in growth media in 96-well microtiter plates at a 100uL volume. Cells were then incubated for 24 hours at 37° C. in ahumidified incubator. The test agent (Plinabulin)'s doses were achievedusing HP D300 Digital Dispenser. Briefly, the digital dispenser addedtest agent (Plinabulin) in a DMSO solvent at precise volumes to mediacontaining wells. Control wells received equivolumes of DMSO that testagent (Plinabulin) wells received. Following addition of the testagents, cells were exposed at the concentrations and dilutions describedin Table 1 for 72 hours at 37° C. in a humidified incubator. Following72 hour exposure, Plinabulin and control media were removed and 200 μlof a 1:1 mixture of media and CellTiter-Glo® Reagent was added to eachwell. The plates were incubated for 60 minutes at 37° C. in a humidifiedincubator. After incubation, luminescence was recorded using aluminometer.

TABLE 1 Single Agent Cell Viability Assay Test Agents Concentrations 1μM high: 10 concentrations; 1:3 dilutions* Standard Agent 1-100 μMhigh¹: 10 concentrations; 1:3 Concentrations dilutions² Assay ExposureTime 72 hours continuous Method CellTiter-Glo ® Cell Viability AssayReplicates 3 replicates per concentration; 2 technical replicates¹Concentrations and dilution factor may be changed after initial screento optimize curve fitting. ²High concentration were selected based onthe expected activity of the respective SOC.

Data were expressed as the percent cell growth of the untreated(vehicle) control calculated from the luminescence signals. Thesurviving fraction of cells was determined by dividing the meanluminescence values of the samples treated with Plinabulin by the meanluminescence values of untreated control. The inhibitory concentration(IC₅₀) value for the Plinabulin treated sample and control wereestimated using Prism 6 software (GraphPad Software, Inc.) bycurve-fitting the data using the non-linear regression analysis.

TABLE 2 Summary of Plinabulin IC₅₀ results for various cancer cell lineswith RAS mutant Cells RAS mutant Tissue Type IC₅₀ (nM) KMS-27 NRAS(p.Q61R) Human Multiple Myeloma 7.84 MOLP-8 NRAS (p.Q61L) Human MultipleMyeloma 4.22 MM.1S KRAS (p.G12A) Human Multiple Myeloma 18.99 L363 NRAS(p.Q61H) Human Multiple Myeloma 9.44 SNG-M KRAS (p.G12V) HumanEndometrial 13.08 HEC1a KRAS (p.G12D) Human Endometrial 57.74 HCT-15KRAS (p.G13D) Human Colorectal 13.08 LoVo KRAS (p.G13D) Human Colorectal6.85 HCT116 KRAS (p.G13D) Human Colorectal 32.75 RXF-393 KRAS (p.D153V)Human Renal 4.37 AsPC-1 KRAS (p.G12D) Human Pancreatic 16.92 Capan-2KRAS (p.G12V) Human Pancreatic 10.33 MIA PaCa-2 KRAS (p.G12C) HumanPancreatic 92.31

As shown in Table 2, Plinabulin as a single active agent has cytotoxiceffects at low IC₅₀ concentrations on various tumor cell lines with aRAS mutation, including both KRAS and NRAS mutations.

Example 2

The combination of Plinabulin and Docetaxel was tested and its activitycompared to the standard chemotherapeutic Docetaxel in the A549 (KRASG12S) human lung tumor xenograft model. The experimental data determinedpotential additive or synergistic effects of Plinabulin in combinationwith Docetaxel in the A549 model.

A549 Human Lung Tumor Xenograft Model

Female nude mice (nu/nu) between 5 and 6 weeks of age weighingapproximately 20 g were obtained from Harlan, Inc. (Madison, Wis.). TheA549 human lung tumor cell line was obtained from the American TypeCulture Collection (ATCC). The A549 human lung tumor cell line hadmutation at KRAS G12S. The tumor originates from an explant culture oflung carcinomatous tissue from a 58 yr old caucasian male. Animals wereimplanted subcutaneously (s.c.) by trocar with fragments of A549 humantumor carcinomas harvested from s.c. growing tumors in nude mice hosts.When the tumors were approximately 46 mg in size (18 days followinginoculation), the animals were pair-matched into treatment and controlgroups. The negative control group contained 8 tumor mice and all othergroups contained 9 tumor mice. Each mouse was ear-tagged and followedindividually throughout the experiment. The study was conducted in anon-GLP setting. Initial doses were given on Day 1 following pairmatching. Plinabulin at 7.5 mg/kg was administered i.p. on two differentschedules—Days 1, 4, 8, 11, and 15 and qd×5 (once dose every day forfive days). 12.5% dimethylsulfoxide (DMSO), 5% cremophor, and 82.5%peanut oil were combined and administered i.p.; qd×5 to serve as thenegative control. Docetaxel (Aventis) was administered i.v. on Days 1,3, and 5 at 12.5 mg/kg was administered i.p.; wkly×3 at 100 mg/kg toserve as the positive control. Plinabulin at 7.5 mg/kg with the qd×5schedule was also administered in combination with Docetaxel at the samedose, route, and schedule as the single agent. In the Plinabulin andDocetaxel combination group, Docetaxel was administered 15-30 minutesprior to Plinabulin.

Plinabulin was weighed, and then 12.5% DMSO, 5% cremophor, and 82.5%peanut oil was added, respectively. Finally, the compounds were mixed byvortex and injected within one hour.

Each group contained eight mice. Mice were weighed twice weekly, andtumor measurements were obtained using calipers twice weekly, startingon Day 1. These tumor measurements were converted to mg tumor weight bythe standard formula, (W2×L)/2. The experiment was terminated when thecontrol group tumor size reached an average of 1 gram. Upon termination,the mice were weighed, sacrificed and their tumors were excised. Thetumors were weighed, and the mean tumor weight per group was calculated.In this model, the mean treated tumor weight/mean control tumorweight×100 was subtracted from 100% to give the tumor growth inhibition(TGI) for each group. One-tailed t-tests using GraphPad Prism© Software(Macintosh version 3.0) were used to calculate p-values.

Some agents may have caused tumor shrinkage in this tumor xenograftmodel. With these agents, the final weight of a given tumor wassubtracted from its own weight at the start of treatment on Day 1. Thisdifference was divided by the initial tumor weight to give the %regression. A mean % tumor regression was calculated from data from micein a group that experienced tumor regressions.

Negative and Positive Controls

The negative control group had a final mean tumor weight on Day 56 of870.4 mg±305.1. Docetaxel (12.5 mg/kg; i.v.; Days 1, 3, 5) served as thepositive control for the study and had a final mean tumor weight of655.1 mg±109.2 and 691.4±175.8, respectively. This resulted in a TGI of26.1% for Docetaxel, compared to the vehicle control group and waswithin the expected activity range seen in past A549 studies performed.There were no toxic deaths observed in the Docetaxel or Irinotecangroups.

Animals in the Docetaxel group experienced some weight loss. On Day 11,initial mean weight loss was recorded at 2.7%. By Day 22, animals hadregained their weight and showed a positive weight gain of 15.4%. Therewere no toxic deaths observed in the positive control group.

Plinabulin and Docetaxel Combination

Intraperitoneal administration of Plinabulin at 7.5 mg/kg on Days 1-5resulted in a mean final tumor weight of 1525.7 mg±355.3. Thecombination group of Plinabulin (7.5 mg/kg; Days 1-5) and Docetaxel(12.5 mg/kg; i.v.; Days 1, 3, 5) had a final mean tumor weight on Day 56of 265.8 mg±113.1. This resulted in a TGI of 74.3%. The combination wassuperior to the Docetaxel treatment group, which had a mean final tumorweight of 655.1 mg±109, and the difference between the combination groupand the Docetaxel single agent group was statistically significant(p<0.05).

Animals in the Plinabulin single agent group experienced weight gain. OnDays 11 and 22, mean weight gain was recorded at 10.6% and 21.8%,respectively. There were no toxic deaths observed in this dose group.Animals in the combination group experienced weight loss. On Day 11,initial mean weight loss was recorded at 2.7%. By Day 22, animals hadregained their weight and showed a 16% weight gain.

At the dose levels and schedules evaluated, animals experienced notoxicity with Plinabulin as a single agent. In all five studies, onlyone death occurred in the Plinabulin group when administered as a singleagent. FIG. 3 shows the tumor weight autopsy in the mice when Plinabulin(7.5 mg/kg) was used in combination with Docetaxel (12.5 mg/kg) throughintraperitoneal route. Plinabulin in combination with Docetaxel againstthe A549 lung tumor model demonstrated a statistically significant(p<0.05) increase in TGI, when compared to Docetaxel alone (74.3% vs.26.1%). The results indicated strong tumor growth inhibition for thecombination of Plinabulin and Docetaxel when compared to Docetaxel as asingle agent, showing potential additive or synergistic effects ofPlinabulin in combination with Docetaxel in the A549 (KRAS G12S) humanlung tumor xenograft model.

Example 3

The mouse model of glioma used was a PDGF-driven GEMM of glioma thatmimics the proneural molecular subgroup of glioblastoma (GBM). Thismodel was based on somatic cell-specific gene transfer; thereplication-competent ALV-splice acceptor (RCAS) retroviral systemallowed the instillation of particular genetic alterations withintightly regulated windows of differentiation in a cell type-specificmanner. The RCAS/tv-a system employed the RCAS retroviral vector toinfect mice genetically engineered to express the RCAS receptor (tv-a)in specific cell populations. Here, gliomas were generated byRCAS-mediated transfer of PDGF to nestin-expressing cells in the brain.Nestin was expressed in a stem/progenitor cell population in the brain,and has been demonstrated to be a marker for cancer stem cells locatedin perivascular regions (PVN) in both human and mouse brain tumors.PDGF-driven gliomas arose with complete penetrance when combined withInk4a-arf−/− deletion by 4-5 weeks post-infection. These tumors closelymimicked the “proneural” subtype of GBM, in which CDKN2A (encoding forboth p16INK4A and p14ARF) deletion was observed in 56% of “proneural”human gliomas. The tumor cell structures that define human gliomas, suchas Scherer structures, microvascular proliferation and pseudopalisadingnecrosis were recreated in this GEMM as shown in FIGS. 1a-1d . Moreover,glioma cells migrated along white matter tracks, surrounded neurons andblood vessels and accumulate at the edge of the brain in the sub-pialspace. In this regard, PDGF-driven GEMMs of glioma closely resemblesPN-GBM, and represents an excellent experimental system to define theinteractions between tumor cells and non-neoplastic cells in the tumormicroenvironment.

The PDGF-induced model of glioma were used to determine response toradiation and temozolomide as shown in FIG. 2. Glioma-bearing mice wereidentified by symptoms and verified by T2 weighted MRI. These mice wereeither treated with vehicle, temozolomide at 25 mg/kg daily for 12 days,or fractionated radiation at a dose of 2 Gy per day 5 days per week for2 weeks (20 Gy total). The top two images in FIG. 2 shows the growth oftumors that were untreated (vehicle); and in contrast, both thetemozolomide treated and irradiated tumors shrank in volume over thatsame period of time. These tumors recurred after treatment and allanimals died of recurrent tumor as can be seen in the survival curvesfor these corresponding cohorts of mice. The testing data for treatmentwith radiation and temozolomide illustrated that: 1) trials wereperformed in this mouse model, 2) the effect of these treatments on micesurvival mirrored the human condition, 3) all the mice died of disease,and 4) the relatively homogenous outcomes of these murine cohortssupported the use of this experimental paradigm to detect survivaldifferences in the study.

PDGF-induced model of glioma prepared using the procedures describedabove were generated. The mice were transgenic for expression of theRCAS receptor (tv-a) from the nestin promoter and having a background ofink4a/arf−/− and lox-stop-lox luciferase, were infected with RCAS-PDGFand RCAS-KRAS that expressed G12D mutant KRAS. The resultant tumorsoccurred within the first 3-4 weeks. The tumors had the histologicalcharacteristics of GBM and can be identified by symptoms of lethargy andpoor grooming, MRI scans using a T2 weighted sequence, orbioluminescence imaging with an IVIS system. Mice in the treatment group(KRAS tumor) were administered i.p. with Plinabulin 7.5 mg/kg two timesper week for 10 wks, and mice in the control group were administeredwith Plinabulin diluent (40% wt Kolliphor® HS15 and 60% wt propyleneglycol) only. These treated mice began to gain weight and show improvedsymptoms within a few days.

Plinabulin was tested on Mice with PDGF-induced gliomas that expressedG12D mutant KRAS. 4-6 week old nestin-tv-a/ink4a-arf−/− mice wereanesthetized with Isoflurane and injected with Df-1 cells transfectedRCAS-PDGF-B-HA, RCAS-KRAS. Mice were injected with one microliter of a1:1 mixture of 2×10⁵ RCAS-PDGF-B-HA/RCAS-KRAS using a stereotactic framevia a 26-gauge needle attached to a Hamilton syringe. Cells wereinjected into the right frontal cortex, coordinates bregma 1.75 mm, Lat−0.5 mm, and a depth of 2 mm. Mice were monitored carefully for weightloss and put on the study when they lost >0.3 grams total over 2consecutive days or displayed outward signs of a tumor. The mice wereentered into a study group and treated with either Plinabulin orPlinabulin diluent as described above while consecutively beingmonitored for lethargy, hunched posture, appetite loss, outward signs oftumor growth, agitation, weight-loss and overall failure to thrive. Themice were sacrificed when they lost more than 20% of their body weight,mobility, inability to feed or weighed less than 14 grams for a male/12grams for a female. The mice were sacrificed using CO₂, brains wereharvested and stored O/N in 10% Neutral buffered formalin and thenreplaced with Flex 80 and stored at 4 degrees.

FIG. 4 shows the survival rate of mice with Glioblastoma with the G12DKras mutation. As shown in FIG. 4, mice having the PDGF-induced model ofGlioblastoma generally had a significantly better survival rate in thePlinabulin treated group as compared to the control group (p=0.001).

Example 4

Mice with PDGF-induced gliomas that expressed G12D mutant KRAS wereprepared using the procedures according to Example 3 and used in thisexperiment. 4-6 week old nestin-tv-a/ink4a-arf−/− mice were anesthetizedwith Isoflurane and injected with Df-1 cells transfected RCAS-PDGF-B-HA,RCAS-KRAS. Mice were injected with one microliter of a 1:1 mixture of2×10⁵ RCAS-PDGF-B-HA/RCAS-KRAS using a stereotactic frame via a 26-gaugeneedle attached to a Hamilton syringe. Cells were injected into theright frontal cortex, coordinates bregma 1.75 mm, Lat −0.5 mm, and adepth of 2 mm. Mice were monitored carefully for weight loss and put onthe study when they lost >0.3 grams total over 2 consecutive days ordisplayed outward signs of a tumor.

The mice were entered into two study groups. One group was treated withthe combination of temozolomide (TMZ), Radiation and Plinabulin:radiation was given at 10 gy×1, TMZ and Plinabulin 7.5 mg/kg inPlinabulin diluent was administered intraperitoneally twice a week onMonday and Thursday for 10 weeks. The other group, the control group,was treated with the combination of TMZ and radiation: radiation wasgiven at 10 gy×1, TMZ was administered intraperitoneally twice a week onMonday and Thursday for 10 weeks. the mice were monitored for lethargy,hunched posture, appetite loss, outward signs of tumor growth,agitation, weight-loss and overall failure to thrive. The mice weresacrificed when they lost more than 20% of their body weight, mobility,inability to feed or weighed less than 14 grams for a male/12 grams fora female. The mice were sacrificed using CO₂; brains were harvested andstored O/N in 10% Neutral buffered formalin and then replaced with Flex80 and stored at 4 degrees. As shown in FIG. 5, the mice having thePDGF-induced model of Glioblastoma generally had significantly bettersurvival rate in the Plinabulin, TMZ, and radiation treated group ascompared to the control group that received TMZ and radiation(p=0.0149).

Example 5

The combination of Plinabulin and irinotecan was tested and its activitywas compared to the standard chemotherapeutic irinotecan in the HCT-15(KRAS mutation G13D; P53 mutation S241F) human colon tumor xenograftmodel. The experimental data determined synergistic effects ofPlinabulin in combination with irinotecan in the HCT-15 model.

HCT-15 Human Colon Tumor Xenograft Model

Female Athymic nude mice (Hsd:Athymic Nude-Foxn1_(nu)) were supplied byHarlan (Indianapolis, Ind.). Mice were received at 4 weeks of age. Allmice were acclimated prior to handling. The HCT-15 human colon tumorcell line was received from ATCC (Manassas, Va.). Cultures weremaintained in RPMI-1640 (Lonza; Walkersville, Md.) supplemented with 10%fetal bovine serum (FBS; Seradigm; Radnor, Pa.), and housed in a 5% CO₂atmosphere. The cultures were expanded in tissue culture flasks at a1:10 split ratio until a sufficient amount of cells were harvested. TheHCT-15 human colon tumor cell line had mutations at KRAS G13D and P53S241F.

Female mice were inoculated in the subcutaneous right flank with 0.1 mlof a 50% Media/50% Matrigel mixture containing a suspension of HCT-15tumor cells (5×10₆ cells/mouse).

Plinabulin (80 mg/20 mL in 40% Solutol:60% propylene glycol) was dilutedin a sterile 5% dextrose solution to a concentration of 0.75 mg/mL.Plinabulin at 7.5 mg/kg was administered i.p. twice weekly until the endof the study. Plinabulin vehicle (40% soltol:60% propylene glycol) wasdiluted in sterile 5% dextrose solution in the same ratio as forplinabulin and was used as a negative control. Irinotecan (TevaPharmaceuticals (Irvine, Calif.) was diluted in 0.9% sodium chloridesolution to a concentration of 10 mg/ml to deliver a dose of 100 mg/kgin a 10 ml/kg dose volume. Irinotecan was dosed intraperitoneally at adose of 100 mg/kg once weekly for three weeks to serve as a positivecontrol. Plinabulin was also administered in combination with irinotecanat the same dose, route, and schedule as the single agent. In thePlinabulin and irinotecan combination group, irinotecan was administered120 minutes prior to Plinabulin

Seven days following inoculation, tumors were measured using a digitalcaliper. The calipers were used to measure width and length diameters ofthe tumor. The measured values were digitally recorded using animalstudy management software, Study Director V.2.1.1 (Study Log). Tumorvolumes were calculated utilizing the formula: Tumor volume(mm₃)=(a×b₂/2) where ‘b’ is the smallest diameter and ‘a’ is the largestdiameters. Forty mice with tumor sizes of 104-125 mm₃ were randomizedinto four groups of ten mice, each with a mean of approximately 112 mm₃,by random equilibration using Study Director (Day 1). Tumor volumes andbody weights were recorded when the mice were randomized and were takentwice weekly thereafter. Clinical observations were made daily.

The experiment was terminated when the control group tumor size reachedan average of 1 gram. Upon termination, the mice were weighed,sacrificed and their tumors were excised. The tumors were weighed, andthe mean tumor weight per group was calculated as well as a visualassessment of tumor necrosis.

Mean tumor growth inhibition (TGI) was calculated for Day 27 (the finalday of the study). All statistical analyses in the xenograft study wereperformed with Prism GraphPad® v6.00 software. Differences in Day 27tumor volumes were confirmed using the Analysis of Variance (ANOVA)test.

Negative and Positive Controls

The negative control group had a final mean tumor volume on Day 27 of1701.4 mm³±178.0. Irinotecan (100 mg/kg; i.p.; Days 1, 8, 15) served asthe positive control for the study and had a final mean tumor volume of1196.2 mm³±121.7. This resulted in a TGI of 31.8% for irinotecan,compared to the vehicle negative control group. There were no toxicdeaths observed in the negative control or positive groups.

Treatment with vehicle negative control, Plinabulin, and irinotecan werewell-tolerated. Some mild body weight loss occurred with the vehiclecontrol group and Plinabulin group in the final few days of the study,but body weight loss was not substantial. Clinical observationsconsisted primarily of tumor necrosis, which is typical for this tumormodel. Combination treatment with Plinabulin and irinotecan was fairlywell-tolerated with moderate body weight loss observed.

The combination group of Plinabulin (7.5 mg/kg; Days 1-5) and irinotecan(100 mg/kg, days 1, 8 and 15) had a final mean tumor volume on Day 27 of766.8 mm³±84.2. This resulted in a TGI of 58.8%. The combination wassuperior to the irinotecan treatment group, which had a mean final tumorvolume of 1196.2 mm³±121.7, and the difference between the combinationgroup and the irinotecan single agent group was statisticallysignificant (p<0.05).

At the dose levels and schedules evaluated, animals experienced notoxicity with Plinabulin as a single agent. FIG. 6 shows the tumorvolume over the course of the study in the mice when Plinabulin (7.5mg/kg) was used in combination with irinotecan (100 mg/kg) via theintraperitoneal route. Plinabulin in combination with irinotecan againstthe HCT-15 colon tumor model demonstrated a statistically significant(p<0.05) increase in TGI, when compared to irinotecan alone (58.8% vs.31.8%). The results indicated strong tumor growth inhibition for thecombination of Plinabulin and irinotecan when compared to irinotecan asa single agent, showing potential additive or synergistic effect ofPlinabulin in combination with irinotecan in the HCT-15 (KRAS G13D)human colon tumor xenograft model.

Example 6

The combination of Plinabulin and irinotecan was tested and its activitywas compared to irinotecan alone in the LoVo (KRAS mutation p.G13D)human colon tumor xenograft model. The experimental data determinedpotential additive or synergistic effects of Plinabulin in combinationwith irinotecan in the LoVo model.

LoVo Human Colon Tumor Xenograft Model: Female Athymic nude mice(Hsd:Athymic Nude-Foxn1_(nu)) were supplied by Harlan (Indianapolis,Ind.). Mice were received at 4 weeks of age. All mice were acclimatedprior to handling. The LoVo human colon tumor cell line was receivedfrom ATCC (Manassas, Va.). Cultures were maintained in F12-K(Corning/Cellgro, Manassas, Va.) supplemented with 10% fetal bovineserum (FBS; Seradigm; Radnor, Pa.), and housed in a 5% CO₂ atmosphere.The cultures were expanded in tissue culture flasks at a 1:3 split ratiountil a sufficient amount of cells were harvested. The LoVo human colontumor cell line had mutations at KRAS p.G13D.

Female mice were inoculated in the subcutaneous right flank with 0.1 mlof a 50% Media/50% Matrigel mixture containing a suspension of LoVotumor cells (1×10⁷ cells/mouse).

Plinabulin (80 mg/20 mL in 40% Solutol:60% propylene glycol) was dilutedin a sterile 5% dextrose solution to a concentration of 0.75 mg/mL.Plinabulin at 7.5 mg/kg was administered i.p. twice weekly until the endof the study. Plinabulin vehicle (40% soltol:60% propylene glycol) wasdiluted in sterile 5% dextrose solution in the same ratio as forplinabulin and was used as a negative control. Irinotecan (TevaPharmaceuticals (Irvine, Calif.) was diluted in 0.9% sodium chloridesolution to a concentration of 10 mg/ml to deliver a dose of 100 mg/kgin a 10 ml/kg dose volume. Irinotecan was dosed intraperitoneally at adose of 80 mg/kg once weekly for three weeks to serve as the positivecontrol. Plinabulin was also administered in combination with irinotecanat the same dose, route, and schedule as each of the single agents. Inthe Plinabulin and Irinotecan combination group, Irinotecan wasadministered 120 minutes prior to Plinabulin.

Seven days following inoculation, tumors were measured using a digitalcaliper. The calipers were used to measure width and length diameters ofthe tumor. The measured values were digitally recorded using animalstudy management software, Study Director V.2.1.1 (Study Log). Tumorvolumes were calculated utilizing the formula: Tumor volume(mm₃)=(a×b₂/2) where ‘b’ is the smallest diameter and ‘a’ is the largestdiameters. Forty mice with tumor sizes of 104-125 mm₃ were randomizedinto four groups of ten mice, each with a mean of approximately 112 mm₃,by random equilibration using Study Director (Day 1). Tumor volumes andbody weights were recorded when the mice were randomized and were takentwice weekly thereafter. Clinical observations were made daily.

The vehicle control was sacrificed after reaching a mean tumor volume ofequal to or greater than 1500 mm₃ on Day 15 as outlined in the protocol.The treatment groups were ended when the irinotecan group reached a meantumor volume of 774 mm₃ due to mice being found dead or moribundsacrificed. At time of necropsy, the tumors were excised and wet weightmeasurements were recorded as well as a visual assessment of tumornecrosis.

Mean tumor growth inhibition (TGI) was calculated for Day 27. Allstatistical analyses in the xenograft study were performed with PrismGraphPad® v6.00 software. Differences in Day 27 tumor volumes wereconfirmed using the Analysis of Variance (ANOVA) test. A one-tailedStudent's T-Test with Welch's correction was also used to verify anydifferences between each group and the vehicle control, and a two-tailedStudent's T-Test was used to verify differences between combinationgroups and their respective single agents.

The Vehicle Control reached a mean tumor volume of 2078.1 mm³ on Day 15.This group experienced no appreciable mean body weight loss throughoutthe study. Nine of ten mice experienced slight to moderate tumornecrosis first observed on Day 12. All animals survived to terminalsacrifice.

Treatment with Plinabulin 7.5 mg/kg resulted in a mean tumor volume of1279.2 mm³ on Day 15. This group produced a TGI of 41.0% (n=7) on Day15. A significant decrease in mean tumor volume was observed (p<0.05)when compared to the vehicle control on Day 15 (Student's T-test). Thisgroup experienced no mean body weight loss throughout the study. Nine often mice experienced slight to severe tumor necrosis first observed onDay 12. Mouse 5 was moribund sacrificed on Day 19 due to clinicalobservations indicating morbidity from severe tumor necrosis. Mouse 2,3, 4, 7, and 10 were found dead during the study. Mouse 8 was found deaddue to technical error. Three out of ten mice survived to terminalsacrifice.

Treatment with irinotecan 80 mg/kg resulted in a mean tumor volume of1156.7 mm³ on Day 15. This group produced a TGI of 47.2% (n=10) whencompared to the vehicle control on Day 15. A significant decrease inmean tumor volume was observed (p<0.05) when compared to the vehiclecontrol on Day 15 (Student's T-test). This group experienced moderatebody weight loss with a maximum of 7.4% on Day 20, the final day of thestudy. Eight of ten mice experienced slight to severe tumor necrosisfirst observed on Day 12. Mouse 3, 4, and 5 were moribund sacrificed onDay 19, 15, and 18 respectively due to clinical observations indicatingmorbidity from severe tumor necrosis. Mouse 1 and 10 were found dead onDay 17 and 20 respectively. Five out of ten mice survived to terminalsacrifice.

Treatment with Plinabulin 7.5 mg/kg and irinotecan 80 mg/kg resulted ina mean tumor volume of 468.4 mm₃ by Day 15. This group produced a TGI of82.4% (n=10) when compared to the vehicle control on Day 15. Asignificant decrease in mean tumor volume was observed (p<0.05) whencompared to the vehicle control on Day 15 (Student's T-test) and singleagent Plinabulin (Student's T-test). No significant difference in meantumor volume was observed when compared to single agent irinotecan onDay 15. This group experienced moderate body weight loss with a maximumof 5.8% on Day 20, the final day of the study. Five of ten miceexperienced slight to moderate tumor necrosis first observed on Day 15.All ten mice survived to terminal sacrifice. As shown in FIG. 7, thecombination of Plinabulin and irinotecan outperformed irinotecan as asingle agent. Due to the loss of mice in the irinotecan alone group, themean tumor volumes decreased from 1157 mm³ on Day 15 to 774 mm³ on Day20, causing the perceived shift in performance.

What is claimed is:
 1. A method of therapeutically treating a cancercharacterized by expressing a mutant form of a RAS protein, comprisingadministering Plinabulin to a subject in need thereof, wherein the RASprotein is a mutant form of a NRAS protein, a HRAS protein, or a KRASprotein comprising one or more amino acid substitutions selected fromthe group consisting of G12C, G12R, G12D, G12V, G12F, G12L, G12N, G13C,G13R, G13S, G13A, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L,Q61R, Q61H, K117N, A146P, A146T, A146V, and D153V.
 2. The method ofclaim 1, wherein the RAS protein is a NRAS protein.
 3. The method ofclaim 1, wherein the RAS protein is a mutant form of a KRAS proteincomprising one or more amino acid substitutions selected from the groupconsisting of G12C, G12R, G12D, G12V, G12F, G12L, G12N, G13C, G13R,G13S, G13A, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R,Q61H, K117N, A146P, A146T, A146V, and D153V.
 4. The method of claim 1,wherein the cancer is selected from colorectal cancer, pancreaticcancer, renal cancer, lung cancer, liver cancer, breast cancer, prostatecancer, gastrointestinal cancer, peritoneal cancer, melanoma,endometrial cancer, ovarian cancer, cervical cancer, uterine carcinoma,bladder cancer, glioblastoma, brain metastases, salivary glandcarcinoma, thyroid cancer, brain cancer, lymphoma, myeloma, and head andneck cancer.
 5. The method of claim 1, wherein the cancer is selectedfrom squamous cell cancer, small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,hepatocellular carcinoma, colon cancer, endometrial carcinoma, andhepatocellular carcinoma.
 6. The method of claim 1, wherein the canceris selected from colorectal cancer, prostate cancer, breast cancer, lungcancer, endometrial cancer, multiple myeloma, pancreatic, renal cancer,and glioblastoma.
 7. The method of claim 6, wherein the cancer isselected from non-small cell lung cancer, pancreatic cancer, andglioblastoma.
 8. The method of claim 7, wherein the cancer is non-smallcell lung cancer.
 9. The method of claim 3, wherein the KRAS proteincomprises mutation at one or more positions selected from codons 12, 13,59, 61, and
 146. 10. The method of claim 3, wherein the mutant form ofthe KRAS protein comprises one or more amino acid substitutions selectedfrom the group consisting of G12C, G12R, G12F, G12L, G12N, G12D, G12V,G13C, G13S, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R,and Q61H.
 11. The method of claim 3, wherein the mutant form of the KRASprotein comprises one or more amino acid substitutions selected from thegroup consisting of G12C, G12R, G12D, G12V, G13C, G13R, G13S, G13A,G13V, A59E, A59G, A59T, Q61K, Q61L, Q61R, Q61H, K117N, A146P, A146T andA146V.
 12. The method of claim 3, wherein the mutant form of the KRASprotein comprises one or more amino acid substitutions selected from thegroup consisting of G12C, G12R, G12D, G12V, G13C, G13R, G13S, G13A,A59E, A59G, A59T, Q61K, Q61L, Q61R and Q61H.
 13. The method of claim 3,wherein the mutant form of the KRAS protein comprises one or more aminoacid substitutions selected from the group consisting of G12C, G12D,G12V, and D153V.
 14. The method of claim 13, wherein the mutant form ofthe KRAS protein comprises one or more amino acid substitutions selectedfrom G12C and D153V.
 15. The method of claim 1, comprising determiningwhether the subject has a KRAS mutation.
 16. The method of claim 2,wherein the NRAS protein comprises mutation at one or more positionsselected from codons 12, 13, 59, 61, and
 146. 17. The method of claim16, wherein the mutant form of the NRAS protein comprises one or moreamino acid substitutions selected from Q61K, Q61H, Q61R, Q61L, Q61N,Q61E, Q61P, A146T, A146P, and A146V.
 18. The method of claim 1,comprising co-administering Plinabulin with an additional active agent.19. The method of claim 18, wherein the additional active agent isDocetaxel and the cancer is human non-small cell lung cancer, or whereinthe additional therapeutic agent is Irinotecan and the cancer is humancolon cancer.
 20. A method of inhibiting proliferation of a cell havinga RAS mutation, comprising contacting the cell with Plinabulin, whereinthe RAS mutation is a mutant form of a NRAS protein, a HRAS protein, ora KRAS protein comprising one or more amino acid substitutions selectedfrom the group consisting of G12C, G12R, G12D, G12V, G12F, G12L, G12N,G13C, G13R, G13S, G13A, G13V, G13P, S17G, P34S, A59E, A59G, A59T, Q61K,Q61L, Q61R, Q61H, K117N, A146P, A146T, A146V, and D153V.
 21. A method ofinhibiting progression of a cancer characterized by expressing a mutantform of a RAS protein in a subject, comprising administering Plinabulinto a subject in need thereof, wherein the RAS protein is a mutant formof a NRAS protein, a HRAS protein, or a KRAS protein comprising one ormore amino acid substitutions selected from the group consisting ofG12C, G12R, G12D, G12V, G12F, G12L, G12N, G13C, G13R, G13S, G13A, G13V,G13P, S17G, P34S, A59E, A59G, A59T, Q61K, Q61L, Q61R, Q61H, K117N,A146P, A146T, A146V, and D153V.